Enterococcus faecalis is a Gram-positive constituent of the gastrointestinal microflora and a significant cause of nosocomial infections. E. faecalis and other enterococci are recalcitrant to many antimicrobial therapies due to innate and acquired resistance mechanisms. Enterococci also have the robust ability to survive harsh conditions and persist in the environment and within a host for prolonged periods. These attributes make enterococcal infections some of the most serious infectious diseases affecting humans. Although several genetic determinants that contribute to E. faecalis virulence have been characterized, the way in which E. faecalis adapts to and persists within a host during the course of infection is unknown. The goal of this application is to elucidate how E. faecalis responds to signals from a mammalian host during infection. The Specific Aims of this application are: 1) Identify the E. faecalis genes that are differentially expressed during experimental infections.;2) Test the role of 3 to 5 selected differentially regulated genes identified in Specific Aim 1 in E. faecalis pathogenesis.;and 3) Characterize the promoter region(s) of 1 to 3 selected genes of interest identified in Specific Aims 1 and 2. Recombinase-based in vivo expression technology and microarray analysis will be used to identify gene targets that are subject to transcriptional regulation during E. faecalis infection in rabbit models of subdermal abscess formation and infective endocarditis. The virulence of strains harboring in-frame deletions of selected genes will be assessed in the endocarditis model. Mapping the transcription start sites of in vivo expressed genes that contribute to endocarditis and developing transcriptional reporter gene fusions to measure promoter activity in vivo will facilitate future analyses of the regulation of these genes. This research will provide insight on how E. faecalis alters its physiology upon sensing the conditions in the host surroundings. The differentially expressed genes identified in this work may be useful targets for novel chemotherapeutic strategies to treat E. faecalis infection. PUBLIC HEALTH RELEVANCE: Enterococcus faecalis, a bacterial species that is frequently associated with antibiotic resistance, is a leading cause of hospital-acquired infections. This application will identify virulence-associated genes that undergo changes in expression during infection in order to gain insight into the ability of the organism to adapt to its surroundings and persist within a host.